Direct Comparative Analysis of a Pharmacogenomics Panel with PacBio Hifi® Long-Read and Illumina Short-Read Sequencing.

BACKGROUND: Pharmacogenetics (PGx) aims to determine genetic signatures that can be used in clinical settings to individualize treatment for each patient, including anti-cancer drugs, anti-psychotics, and painkillers. Taken together, a better understanding of the impacts of genetic variants on the corresponding protein function or expression permits the prediction of the pharmacological response: responders, non-responders, and those with adverse drug reactions (ADRs). OBJECTIVE: This work provides a comparison between innovative long-read sequencing (LRS) and short-read sequencing (SRS) techniques. METHODS AND MATERIALS: The gene panel captured using PacBio HiFi® sequencing was tested on thirteen clinical samples on GENTYANE's platform. SRS, using a comprehensive pharmacogenetics panel, was performed in routine settings at the Civil Hospitals of Lyon. We focused on complex regions analysis, including copy number variations (CNVs), structural variants, repeated regions, and phasing-haplotyping for three key pharmacogenes: CYP2D6, UGT1A1, and NAT2. RESULTS: Variants and the corresponding expected star (*) alleles were reported. Although only 38.4% concordance was found for haplotype determination and 61.5% for diplotype, this did not affect the metabolism scoring. A better accuracy of LRS was obtained for the detection of the CYP2D6*5 haplotype in the presence of the duplicated wild-type CYP2D6*2 form. A total concordance was performed for UGT1A1 TA repeat detection. Direct phasing using the LRS approach allowed us to correct certain NAT2 profiles. CONCLUSIONS: Combining an optimized variant-calling pipeline and with direct phasing analysis, LRS is a robust technique for PGx analysis that can minimize the risk of mis-haplotyping.

Circulating H3K27 Methylated Nucleosome Plasma Concentration: Synergistic Information with Circulating Tumor DNA Molecular Profiling.

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment.

Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays.

INTRODUCTION: Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. PATIENTS AND METHODS: Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. RESULTS: In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. CONCLUSIONS: The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

MagPure chip: an immunomagnetic-based microfluidic device for high purification of circulating tumor cells from liquid biopsies.

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.

Embryonated Chicken Tumor Xenografts Derived from Circulating Tumor Cells as a Relevant Model to Study Metastatic Dissemination: A Proof of Concept.

Patient-Derived Xenografts (PDXs) in the Chorioallantoic Membrane (CAM) are a representative model for studying human tumors. Circulating Tumor Cells (CTCs) are involved in cancer dissemination and treatment resistance mechanisms. To facilitate research and deep analysis of these few cells, significant efforts were made to expand them. We evaluated here whether the isolation of fresh CTCs from patients with metastatic cancers could provide a reliable tumor model after a CAM xenograft. We enrolled 35 patients, with breast, prostate, or lung metastatic cancers. We performed microfluidic-based CTC enrichment. After 48-72 h of culture, the CTCs were engrafted onto the CAM of embryonated chicken eggs at day 9 of embryonic development (EDD9). The tumors were resected 9 days after engraftment and histopathological, immunochemical, and genomic analyses were performed. We obtained in ovo tumors for 61% of the patients. Dedifferentiated small tumors with spindle-shaped cells were observed. The epithelial-to-mesenchymal transition of CTCs could explain this phenotype. Beyond the feasibility of NGS in this model, we have highlighted a genomic concordance between the in ovo tumor and the original patient's tumor for constitutional polymorphism and somatic alteration in one patient. Alu DNA sequences were detected in the chicken embryo's distant organs, supporting the idea of dedifferentiated cells with aggressive behavior. To our knowledge, we performed the first chicken CAM CTC-derived xenografts with NGS analysis and evidence of CTC dissemination in the chicken embryo.

Self-Assembled Permanent Micro-Magnets in a Polymer-Based Microfluidic Device for Magnetic Cell Sorting.

Magnetophoresis-based microfluidic devices offer simple and reliable manipulation of micro-scale objects and provide a large panel of applications, from selective trapping to high-throughput sorting. However, the fabrication and integration of micro-scale magnets in microsystems involve complex and expensive processes. Here we report on an inexpensive and easy-to-handle fabrication process of micrometer-scale permanent magnets, based on the self-organization of NdFeB particles in a polymer matrix (polydimethylsiloxane, PDMS). A study of the inner structure by X-ray tomography revealed a chain-like organization of the particles leading to an array of hard magnetic microstructures with a mean diameter of 4 µm. The magnetic performance of the self-assembled micro-magnets was first estimated by COMSOL simulations. The micro-magnets were then integrated into a microfluidic device where they act as micro-traps. The magnetic forces exerted by the micro-magnets on superparamagnetic beads were measured by colloidal probe atomic force microscopy (AFM) and in operando in the microfluidic system. Forces as high as several nanonewtons were reached. Adding an external millimeter-sized magnet allowed target magnetization and the interaction range to be increased. Then, the integrated micro-magnets were used to study the magnetophoretic trapping efficiency of magnetic beads, providing efficiencies of 100% at 0.5 mL/h and 75% at 1 mL/h. Finally, the micro-magnets were implemented for cell sorting by performing white blood cell depletion.

Routine Molecular Screening of Patients with Advanced Non-SmallCell Lung Cancer in Circulating Cell-Free DNA at Diagnosis and During Progression Using OncoBEAMTM EGFR V2 and NGS Technologies.

BACKGROUND AND OBJECTIVES: The use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors. METHODS: We present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540). RESULTS: The sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500-3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5-0.1%, and 0.1-0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs. CONCLUSIONS: The OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments.

cfDNA in pancreatic neuroendocrine carcinoma management with Cushing's syndrome.

Summary: We report a case of metastatic pancreatic neuroendocrine carcinoma associated with paraneoplastic Cushing's syndrome, successively treated with five lines of treatment (platin-etoposide, LV5FU2-dacarbazine, FOLFIRINOX, pembrolizumab, and paclitaxel) and anti-secretory treatment. Circulating-free DNA (cfDNA) was analysed at each morphological evaluation starting from the second-line treatment. cfDNA changes were well correlated with the disease course, and cfDNA may be used as a predictive marker and/or as an early marker of response. In addition, the absolute count of atypical cells was elevated upon disease progression. Learning points: cfDNA changes were well correlated with the Cushing's syndrome course and with the tumour burden changes assessed by laboratory markers and by RECIST criteria.cfDNA analysis was used to determine the pharmacogenetic patterns of the present patient.An elevated number of atypical circulating cells was noticed upon disease progression.

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1-10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in future application in mimicking circulating tumor cells.

Cell-free plasma DNA (cfDNA) and mimicking circulating tumor cells (mCTCs) have demonstrated tremendous potential for molecular diagnosis of cancer and have been rapidly implemented in specific settings. However, widespread clinical adoption still faces some obstacles. The purpose was to compare the performance of a BEAMing (beads, emulsion, amplification, and magnetics) assay (OncoBEAM™-epidermal growth factor receptor [EGFR] [Sysmex Inostics]) and a next-generation sequencing assay (NGS; 56G Oncology panel kit, Swift Bioscience) to detect the p.T790M EGFR mutation in cfDNA of non-small cell lung cancer (NSCLC) patients. CfDNA samples (n = 183) were collected within our hospital from patients having a known EGFR sensitizing mutation, and presenting disease progression while under first-line therapy. EGFR mutations were detected using NGS in 42.1% of samples during progression in cfDNA. Testing using the OncoBEAM™-EGFR assay enabled detection of the p.T790M EGFR mutation in 40/183 NSCLC patients (21.8%) versus 20/183 (10.9%), using the NGS assay. Samples that were only positive with the OncoBEAM™-EGFR assay had lower mutant allelic fractions (Mean = 0.1304%; SD ± 0.1463%). In addition, we investigated the detection of p.T790M in mCTCs using H1975 cells. These cells spiked into whole blood were enriched using the ClearCellFX1 microfluidic device. Using the OncoBEAM™-EGFR assay, p.T790M was detected in as few as 1.33 tumoral cells/mL. Overall, these findings highlight the value of using the OncoBEAM™-EGFR to optimize detection of the p.T790M mutation, as well as the complementary clinical value that each of the mutation detection assay offers: NGS enabled the detection of mutations in other oncogenes that may be relevant to secondary resistance mechanisms, whereas the OncoBEAM™-EGFR assay achieved higher sensitivity for detection of clinically actionable mutations.